Lab. No.1

Microbiology:Microbiology: It is the science that deals with It is the science that deals with

the study of microthe study of micro--organisms organisms (very small organisms) that are (very small organisms) that are invisible to the naked eyeinvisible to the naked eye

BacteriaBacteria

VirusesViruses

Fungi Fungi

ProtozoaProtozoa

Pathogenic micro-orgnismsPathogenic micro-orgnisms::

1- Macroscopical Examination 1- Macroscopical Examination (colony morphology):(colony morphology):• Characters of coloniesCharacters of colonies..• HemolysisHemolysis on blood agar. on blood agar.• PigmentPigment production. production.

2- Microscopical Examination:2- Microscopical Examination:• Examination of Examination of wet mount preparationwet mount preparation..• Examination of Examination of stained preparation.stained preparation.

3-Biochemical Tests:3-Biochemical Tests: ((The ability to attack various substances The ability to attack various substances e.g., carbohydrate breakdown;e.g., carbohydrate breakdown;or to produce particular metabolic products or to produce particular metabolic products e.g., enzymes.e.g., enzymes.

4-Additional Tests:4-Additional Tests: such as such as seriological testsseriological tests

Bacteria Gram’s Stain

Gram’s +ve Gram’s -ve

Cocci Bacilli Cocci Bacilli

1. Microscopical Appearance:(Gram’s Stain)1. Microscopical Appearance:(Gram’s Stain)

Gram’s +ve Cocci

Irregular Clusters Chains or PairsTetrads

Staphylococci Micrococci Streptococci

2-Bichemical reactions:2-Bichemical reactions:

1- catalase test.1- catalase test.

2- O/F Test (Oxidation FermentationTest).2- O/F Test (Oxidation FermentationTest).

1- Catalase Test1- Catalase TestDifferentiative test to separateDifferentiative test to separate Staphylococci and Staphylococci and Micrococci which are catalase +veMicrococci which are catalase +ve from from Sterptococci which are catalase –ve.Sterptococci which are catalase –ve.

H2O2

Catalase enzymeH2o + O2

Air bubbles

Principle:

Procedure:

1

3H2O2

2

Results:

Positive test: rapid appearance of gas bubbles.

Staphylococci or Micrococci

Catalase –ve

Streptococci

Catalase +ve

Gram’s +ve Cocci

Irregular Clusters Chains or PairsTetrads

Staphylococci Micrococci Streptococci

Catalase +ve Catalase -ve

2- O/F Test (Oxidation FermentationTest)2- O/F Test (Oxidation FermentationTest)

To differentiate between To differentiate between Staphylococci Staphylococci andand Micrococci. Micrococci.

Principle: Saccharolytic bacteria attack carbohydrates either:

• fermentatively ( in absence of oxygen) to yield relatively strong acids, or

• oxidatively ( in presence of oxygen) to yield weak acids.

Oxidation process is much more easier than Fermentation process.

Procedure:

O F

1 ml liquid paraffin

O-/F- O+/F+ O+/F- O-/F+

Results:

Staphylococci Micrococci

Fermentative OxidativeNon Saccharolytic

Positive Test:

Gram’s +ve Cocci

Irregular Clusters Chains or PairsTetrads

Staphylococci Micrococci Streptococci

Catalase +ve Catalase -veO+/F+ O+/F-

1- Microscopical Examination (Morphology):

2- Macroscopical Examination(cultural characteristics):

No special requirements for growth→ grow on simple nutrient media.On nutrient agar → most strains of S.aureus produce golden yellow colonies.

Gram’s +ve cocci arranged inirregular clusters, non motile, non-sporeforming.

1.1. Coagulase Test.Coagulase Test.

2. Mannitol Fermentation Test.2. Mannitol Fermentation Test.

3. Deoxyribonuclease (DNase) Test3. Deoxyribonuclease (DNase) Test

3- Biochemical reactions:3- Biochemical reactions:

Definitive test to differentiate between Definitive test to differentiate between S.aureusS.aureus & & other species of staphylococci (coagulase-other species of staphylococci (coagulase-negative staphylococci “CONS”)e.g. negative staphylococci “CONS”)e.g. S.epidermidisS.epidermidis

1. Coagulase Test:1. Coagulase Test:

Principle:

FibrinogenPlasma

Coagulase enzymeFibrin

Visible Clot

Procedure:2

3 Place at water bath at 37oC, observe for formation of visible clot for up to 4 hrs.

1

Results:Positive test: formation of visible clot.

Coagulase +ve

S.aureus

Coagulase -ve

S.epidermidis

Mannitol salt agar Mannitol salt agar is a selective medium for is a selective medium for Staphylococcus speciesStaphylococcus species (contains high conc. of salt (contains high conc. of salt (about 7.5%).(about 7.5%).

It is also a differential medium forIt is also a differential medium for S.aureus S.aureus which is the which is the only species of staphylococci that can ferment only species of staphylococci that can ferment mannitol→ acid production → yellow color around the mannitol→ acid production → yellow color around the growth (due to change the color of the pH indicator).growth (due to change the color of the pH indicator).

N.B:N.B:Test sugar: mannitolTest sugar: mannitolpH indicator: phenol red (red in alkaline, yellow in acidic pH indicator: phenol red (red in alkaline, yellow in acidic pH).pH).

2. Mannitol Fermentation Test:2. Mannitol Fermentation Test:

Procedure:

1. Inoculate MSA plate with the test organism by streaking.

2. Incubate the plate at 35oC for 24 hrs.

Flam & Cool

Flam & Cool

Flam & Cool

Mannitol fermentation is indicated by formation of yellow colour around the growth

Growth with yellow colour around the colonies S.aureus

Growth without change in the colour of the medium CONS

Results:

3. Deoxyribonuclease (DNase) Test:3. Deoxyribonuclease (DNase) Test:

Principle:

DNADNase enzyme

NucleotidesInsoluble

In acidsoluble In acid

Procedure:1. Inoculate DNase agar plate with the test organism.

2. Incubate the plate at 35oC for 24 hrs.

3. Flood the plate with 1M HCl.

Results:

DNase activity is indicated by a clear zone around the growth after addition of Hcl

Clear zone around the growth while the rest of the plate appears cloudy

S.aureus

Cloudiness in all the plate

S.epidermidis

1- Penicillin-resistant 1- Penicillin-resistant S.aureusS.aureus:: 95% of 95% of S.aureus & S.epidermidis S.aureus & S.epidermidis

strains are resistant to penicillin. strains are resistant to penicillin.

ttt:ttt: a- semisynthetic penicillins a- semisynthetic penicillins (penicillinase-stable)e.g., (penicillinase-stable)e.g., oxacillin & oxacillin & methicillin.methicillin.

b- b- cephalosporinscephalosporins..

2- methicillin-resistant S.aureus (MRSA):2- methicillin-resistant S.aureus (MRSA):

ttt: ttt: vancomycin.vancomycin.

3- Vancomycin-resistant 3- Vancomycin-resistant S.aureusS.aureus (VISA/ (VISA/ VRSA):VRSA):

ttt: ttt: Linezolid.Linezolid.

Practical WorkPractical Work

Gram’s Stain (spots).Gram’s Stain (spots).

Catalase test.Catalase test.

O/F test.O/F test.

Coagulase test.Coagulase test.

MSA test.MSA test.

DNase test.DNase test.

ResultsResultsMicrococciMicrococci S.aureusS.aureus S.epidermidisS.epidermidis

Gram’s StainGram’s Stain

Catalase Catalase testtest

+ve+ve(air bubbles)(air bubbles)

+ve+ve +ve+ve

O/F testO/F test OO++/F/F-- OO++/F/F++ OO++/F/F++

Coagulase Coagulase testtest

-- +ve+ve

(Visible clot)(Visible clot)

-ve-ve

(No clot)(No clot)

MSA testMSA test -- Growth with Growth with change in colour change in colour into yellowinto yellow

Growth without Growth without any change in any change in colourcolour

DNase testDNase test -- Clear zone around Clear zone around the growth while the growth while the rest of the the rest of the plate is turbidplate is turbid

Turbidity in all the Turbidity in all the plateplate

Gram’s +ve CocciTetrads Clusters Clusters